Diversity of Striated Muscle. Department of Biology, University of Oregon, Eugene 97403

Transcription

1 AM. ZOOLOGIST, 7: (1967). Diversity of Striated Muscle GRAHAM HOYLE Department of Biology, University of Oregon, Eugene SYNOPSIS. A broad comparative survey has been made correlating ultrastructure of cross-striated fibers with contractile properties in both invertebrates and vertebrates. Most of the muscles were found to be heterogeneous in fiber-composition as indicated by: length of sarcomere, extent of SR, number of invaginating tubules, numbers of mitochondria, etc. Z discs and M bands have markedly different structures in different fibers. The general concept of the "fibrillar" nature of striated muscle is challenged. It is suggested that following excitation the responses of individual sarcomeres, or parts of sarcomeres, are relatively independent. The possibility that all striated muscles contain a very thin elastic filament in parallel with actin and niyosin, which may also be contractile, is raised. There have been a great many symposia on muscle in recent years, largely aimed at building up a picture of the structure and function o a hypothetical, generalized, functional unit. As a result, such a general picture has emerged. The purpose of the present symposium is to examine some of the rich diversity to be found in the muscles of a variety of organisms, both vertebrate and invertebrate, with a view both to illuminating the body of "basic" knowledge and also critically evaluating it. If it is truly universal it should be possible to understand the diversity as variations on the theme. But if there are serious discrepancies the comparative physiologist should question the validity of the general model. We may recognize five principal aspects of muscle: ultrastructure and molecular architecture, chemistry, dynamics, neural control, and contraction-coupling. The field of muscle chemistry is a specialized, complex one which, unfortunately, is still somewhat remote from contact with other aspects. For the most part it has been omitted from the topics to be presented at this meeting. With the exception of molluscan muscles, which have been of great interest historically to comparative physiologists, our attention will be confined largely to striated muscles, We shall first consider the ultrastructural components Supported by research grants: GB 3160 from National Science Foundation and NBO from the U. S. Public Health Service. (435) and molecular architecture of a variety of striated muscles and the light this throws on the molecular mechanism of contraction (Aubert, Jean Hanson, Reedy, Pat Mc- Neill, Walcott, Ridgway). Next, continuing to pursue lines which have been of great interest to comparative physiologists, arthropod neuromuscular mechanisms will be reviewed by Atwood and Usherwood. Peachey and Smith will consider morphological features related to excitation-contraction coupling. Molluscan muscle will be covered by Millman and Betty Twarog. Reuben, Ashley, Selverston and Edwards will consider physiological aspects of excitation-contraction coupling. Problems of dynamics in heart muscle will be reviewed by Brady, and giant barnacle fibers by Abbott and myself. There are surprising similarities between the development of active state in some invertebrate skeletal muscles and vertebrate heart muscles, which do not follow the classical model of A.V. Hill. Baskin will give an account of work on changes in volume during contraction and Pringle and Abbott have kindly agreed to present overviews and criticism in a formal way. A link to littleconsidered aspects of muscle chemistry will be made through Van der Kloot. In our laboratory in Eugene, Oregon, and at the marine laboratories in Friday Harbor, Washington, and Coconut Island, Hawaii, we have been making correlated ultrastructural and physiological studies on

2 436 GRAHAM HOYI.E a variety of striated muscles. So far we have examined the following: VERTEBRATE rabbit, psoas, sartorius; garter snake, "slow" body-wall, "fast" bodywall; frog sartorius, gastrocnemius; gecko, sartorius, toe; fish, main fin. INVERTEBRATE crab, Cancer magister, accessory flexor, extensor of dactyl "fast", "slow", intermediate fibers; crab, Podophthalmus, eyestalk levator white and pink fibers; crab, Paralilhoides, leg; crab, Partunus, leg "fast", "slow", various intermediate fibers; squid, Loligo, mantle; insect, Benacus, flight, siphon retractor; insect, Scltistocerca, extensor tibiae, spiracular closing, anterior coxal adductor; insect, Periplaneta, extensor tibiae; copepod, Doropygus seclusus, antennal; barnacle, Balanus nubilusj depressors white and pink fibers, adductors. The task of describing all this material in a formal way is gargantuan and presents formidable difficulties in publication. 1 will refer to it in this introductory talk only in a general way, drawing upon selected examples from our experience in an attempt to give an overall picture and to raise questions. A total of five matters relating- to our generalized concept of structure and function of striated muscle will be considered. The first point to raise is our growing awareness of the inhomogeneity of muscles. In cold-blooded vertebrates (Kuffler and Vaughan Williams, 1953) and birds (Ginsborg, i960) it is now well-established that muscles may contain specialized "slow"' and "fast" fibers. This has not yet been clearly established for mammals, except possibly for intrinsic eye muscles (Hess and Pilar, 1963), although here the "slow" fibers are perhaps not strictly comparable to frog "slow" fibers. However, there are marked differences in the lipid-staining reactions of individual mammalian fibers (Gauthier and Padykula, 1965; George and Susheela, 1961) as well as in invertebrates (George and Bhakthan, 1961). Some are apparently enzymatically lipolytic, while their immediate neighbors utilize only carbohydrate. Others are intermediate. There are probably associated gross physiological differences, resulting in effectively "fast"- phasic, "slow"-tonic, and intermediate forms. Such differences have been positively established by detailed analysis of single fibers in several crustacean muscles (Atwood, this symposium; Hoyle, 1967, in preparation; Atwood, et ah, 1965). In the latter, we now have examples of muscles in which the different kinds of fiber occur mixed together (extensor muscles of walking legs) and others in which they are segregated into distinct bundles (accessory flexor Cancer; anterior rotator of paddle Portunus; levator of eyestalk Podophthalmus). The different fibers may be dramatically different in regard to length of sarcomere. For example, in a minute copepod muscle comprising three or four fibers, one fiber has a sarcomere length of 4 yu., while its neighbors are 12 ^ (Fig. la). Such fibers have twitch times which are roughly proportional to the logarithm of the length of the A band: 40 ms, 200 ms, and 600 ms, respectively (Hoyle, in preparation). The twitch of the whole muscle is a statistical sum, in this case a simple one, but enormously complex in a larger muscle. Another example of the kinds of differences we are beginning to find between adjacent fibers is shown in Fig. lb. The muscle is the extensor tibiae of Periplaneta americana. There are several conspicuous differences, but the most notable is in regard to thin filaments, which are very numerous in one fiber, but sparse in the other. The various portions of a large, complex muscle may be separately innervated and function independently. Or they may receive branches from the same axon and function together (Dorai-Raj, 1964). However, the different fibers can still function to some extent independently, as a result of different pattern sensitivities of their neuromuscular junctions. The smallest striated fibers occur in coelenterates and are about 1 p in diameter. The largest ones occur in giant barnacles and king crabs, reaching a maximum of 5 mm in diameter. There are intrinsic problems in the excitation of such large fibers, which

3 DIVERSITY OF STRIATED MUSCLE 437 FIG. la,b. Differences in adjacent fibers in small muscles, a. Longitudinal section through a small copepod muscle containing two, possibly three, different kinds of muscle fiber in the same very small muscle. Magnification about X The narrow sarcomeres are 4 /i long, the wide ones about 12 p. Note different widths of Z discs; clear H zones are present in short sarcomere fibers, but not in long sarcomere ones. Micrograph courtesy ot Dr. W. H. will be discussed by Selverston and Peachey. Turning our attention now to the details of individual fibers, we find it necessary to question the universal use of the term "fibril" to designate a discrete sub-unit of a fiber. Some "Fibrillenstruktur" fibers appear in transverse section to be divided neatly into clusters of myofilaments each surrounded completely by sarcoplasmic reticulum (SR). A close inspection, however, always reveals one or a few gaps in the continuity of this sarcoplasmic reticular "skin" (Fig. 2). Even if one particular section does not reveal such a gap for any given cluster, another section farther along the same sarcomere is very likely to do so. Furthermore, if we attempt to trace the Fahrenbach. b, Transverse section through two muscle fibers in extensor tibiae of Periplaneta americana. One has a large number of actin filaments, the other very few. There are also differences in the SR. Some clusters of actin filaments stand alone (arrows). Neither diads nor transverse tubules may be found in the fiber having few thin filaments. X 42,000. same "fibril" for more than a few sarcomeres we find that we cannot do so. Over a few segments the SR envelope comes to envelop quite different sets of filaments. Insect "fibrillar" flight muscle may be the only exception. At the other extreme, in some "Feklerstruktur" fibers, there is very little SR at all (Hess, 1965; Hoyle, et al, 1966). All kinds of intermediates exist in some mixed muscles. Some fibers have long, radiating strips of SR, making the fiber look like the wheel of a sports-car. It is clear that striated muscle in general is better considered as being composed of a mass of myofilaments which is invaded to a greater or lesser extent by sarcoplasmic.

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5 DIVERSITY OF STRIATED MUSCLE 439 FIG. 2. Trans\erse sections through tspical vertebrate striated muscle fibers. Xote that adjacent "fibrils" are connected to each other by bridges, a, Fish tail muscle (Tramatomus bernachii Boulanger). Note that adjacent sarcomeres are not in line, the section passing through two H zones, three overlap zones, and two I bands, b, Rabbit psoas. FIG. 3. Nature of Z discs, a. Fish tail muscle (T. bernacchii) showing "classical" zig-zag arrangement of linkage between actin filaments from adjacent sarcomeres, densely-staining, b, Garter snake, ribs to body wall. Overlapping arrangement with lateral cross-connections, c, Copepod larva, anten- FIG. 4. Nature of M bands, a, Rabbit psoas. Three or five parallel sets of bridges are visible. Note also the complex, overlapping nature of the Z disc, b, Insect (Benacus), fibrillar flight muscle. Five to seven parallel sets of bridges, c, Locust (Schistocerca), extensor tibiae. Wide sarcomere at rest here but slightly stretched. The whole region shown is the H zone. Cross connections occur randomly over the whole region, d, Crab (Podo- FIG. 5. Arrangements of thin and thick filaments, a, Garter snake. 2:1 ratio approximates classical model, but is somewhat loose, b, Insect flight (Benacus). 3:1 ratio, extremely regular arrangement. Thick filaments have a double ring structure, c, Rabbit psoas, classical arrangement, d, Copepod larva, antennulary muscle. 3:1 ratio resembles insect flight muscle, e, Locust (Schistocerca), FIG. 6. Longitudinal section through ribs-skin muscle fiber of garter snake fixed in a state of isometric contraction. Note the uneven nature of reticulum, which is anastomosing in three dimensions. When the SR is very extensive, a superficial appearance is given in transverse sections of a complete division. My second consideration is the nature of Z discs (Kxause's "membranes"). These are still referred to in important articles as "membrar.es" (Huxley, 1965) and even assigned a significant functional role as conductors of excitation inwards (Garamvolgi, 1963). It is now abundantly clear, however, that no known Z disc resembles, in its fine structure, a cellular element which a membrane physiologist would recognize as a source of ionic separation. Also, classical experiments have demonstrated that oil droplets pass very freely through Z discs. Secondly, Z discs come in great variety. Some are thin, others five times as thick. Here the arrangement of filaments in the overlap /one is aupical. M\osiiv> are ver\ thick and surrounded by large, irregular groups of thin filaments. A section through a Z disc is included, showing the square, window-field arrangement of filaments. X 30,000. nular muscle. Very little structure and no density. Some thin filaments are cross-linked, others appear to go straight across, d, Locust (Schistocerca), extensor tibiae. Alternating attachments of thin filaments, but broad band with numerous cross-connections. X 120,000. phthalmut), white eye-raising fiber. Center of the H zone is bare; two zones of cross-bridges occur, each in rows of 3-5. e, Cross-section of M band of fish tail muscle, showing connecting bridges, f, Cross-section of H zone of stretched depressor fiber of barnacle (B. nubilus). Random cross-connections; these probably occur throughout the A band. All X 80,000. jumping leg. 4:1 ratio (orbits of 10). f, Crab (Podophthalmus), pink eye-raiser fiber. 7:1 ratio. No regular orbits of thin filaments. The fiber was stretched and showed H zones; the high ratio and ii regular arrangement are natural and do not represent the result of a double overlap of thin filaments. All X 100,000. 5b, courtesy of B. Walcott. contractions of individual sarcomeres or rather parts of half-sarcomeres (the basic functional unit). Some are shortened, others extended. X 12,000. Patricia Dudley has found very fast-contracting, beautifully-striated muscles in a copepod larva which completely lack any dense Z line in most of their sarcomeres (Fig. 3c). We have found that lipid solvents readily remove the density from the Z discs in vertebrate fibers. What is left closely resembles the appearance of the I band of the copepod fibers in the relevant Z region, namely, a set of fine filaments, some of which appear to be thinner than actin. In some Z discs, there is a zig-zag appearance in which alternate actins from contiguous sarcomeres are cross-connected by thinner filaments which may be single actin strands, or another material (Fig. 3a). Such discs have a "basket-weave" appearance in transverse section {e.g., Reedy,

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7 DIVERSITY OF STRIATED MUSCLE , who suggested the term). Other discs show an overlap of actin filaments or of extensions from them, which are thinner than actins (Fig. 3b). Discs of this kind are crossconnected by fine filaments in a simple square array, forming a "window" pattern in transverse section. Yet other discs seem to be composed only of a dense mass of fine filaments running longitudinally, together with many fine lateral connections (Fig. 3d). A third question concerns the means of linking thick filaments together. Some muscles, such as frog and fish, have a set of three to five rows of cross-bridges located exactly in the center of the H zone forming the M band region (Fig. 4a). Insect flight muscle is similar, but has more rows of bridges. In transverse section they present a neat hexagonal pattern (Fig. 4e). However, in other muscles such as the copepod antennal and Podophthalmns eye-raiser, the center of the sarcomere is clear but there is a double M band, comprised of two sets of bridges, one on each side (Fig. 4d). Cross-connections between the myosin filaments occur at many sites, possibly along their entire length (Fig. 4f). The fourth issue is the relationship of actin to myosin filaments. The first fibers in which the filaments were clearly seen were from rabbit psoas and blowhy flight muscle. The former have a 2:1 actin/myosin ratio, the latter a 3:1 ratio (Fig. 5a, b). In each case the actins are in neat orbits. Such an array has been considered to be associated intimately with the mechanism of contraction. Now we can add fibers to the list in which the ratio is 4:1 providing orbits of 10 (locust extensor tibiae Fig. 5e), and 5:1 (several insect and crab muscles) providing orbits of 12 (see also Auber, this symposium). However, the orbits are by no means neat and regular when they are also large. Some contain as few as 4 thin filaments, others as many at 20. To the list we can add a special kind of fiber found in the eyestalk levator muscle of Podophthalmus in which there is a 7:1 ratio of thin to thick (Fig. 5f). Here there is virtually no orbiting. The actins form small clusters of 6-40 filaments arranged irregularly, or in places in a "square" array, while the myosins fall very irregularly between them and may even have no actins around them. Thus, a neat array is not an essential feature of the molecular architecture of a striated muscle fiber. The fifth point concerns the dogma that striated muscle contains only two longitudinally-arranged filamentous proteins, actin and myosin. Upon this belief rests the need to invoke cross-bridges between them as the basis for both development of tension and changes in length. How firmly established in this generalization? Muscles do not fall apart, nor do they show any sudden fall in stiffness when stretched to just beyond the point of overlap. This could be because the elastic strength of the sarcolemma, and perhaps also the SR, hold the contractile apparatus together. However, fibers from which the sarcolemma has been stripped behave in a similar manner, and Hanson and Huxley (1956) have reported that single rabbit fibrils from which the myosin had been removed may remain intact and are elastic. Allen Selverston, in our lab, was able to stretch a split glycerinated fibril from Balarms niibilns in the presence of ATP, to well beyond the overlap point (Fig. 7). There was no chance in this experiment that SR could be holding the material together. Of considerable interest is the fact that the sarcomeres did not stretch equally. Some retained almost rest length, while others reached about 3X rest length. The latter became quite thin, yet still did not break. Such fibrils return to their original shape. Electron micrographs made of similar stretched material show that although the A-band of heavily-stretched sarcomeres is somewhat elongated due to a partial separation of filaments (they are displaced but not stretched), there is a marked gap between it and the T-band filaments at both ends (Fig. 8). There was also a gap in some instances between the Z disc and the 1 filaments. The gaps must be bridged by fine elastic filaments. In their original proposal regarding the

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12 441 GRAHAM HOYLE filament composition of striated muscle, Hanson and Huxley (1956) proposed a hypothetical elastic, very thin filament, which they labelled the S filament, joining the ends of the actin filaments across the H zone. They also proposed a similar unnamed filament linking the other ends of the actin to the Z discs. Both filaments were dropped from their definitive accounts, however. I would like to propose, not that we return to this model, but rather that we replace it by a different one with which their earlier suggestions are simply compatible. On this model there is a third filament, which I propose to call the T filament, extending from one end of the fiber to the other, passing through Z discs and H zones alike (Fig. 9). T may stand for very thin. The T filament must be highly elastic, and therefore must play an important passive role in muscle. The length of the series elastic component in barnacle muscles, as determined at the peak of contractibility, is about 14% of the total rest length. This is so long that it can only be explained on the basis that it is provided by a component of the sarcomeres. This finding, together with the end-to-end location and undoubted sti-ength of the T filament, suggests the possibility that it is also concerned with contraction. We have embarked upon a series of investigations aimed at attempting to identify T filaments in various muscles. Some of these will be described by my colleagues, Patricia McNeill, Benjamin Walcott, and Ellis Ridgway. Suffice to say that I am now satisfied that such a filament exists, although the demonstration is very difficult in view of its extreme thinness. In regard to its possible contractibility, all attempts to show that contraction fails at the point of non-overlap, which is re- FIG. 7. Single split myofibril from glycerinated lateral depressor of B. nubilus stretched in the presence of ATP. Note the uneven nature of the changes in length of sarcomere. Three sarcomeres are stretched beyond the overlap point of actin and myosin filaments, yet do not break (see electron micrographs in paper by McNeill and Hoyle). This micrograph, courtesy of A. Selverston.

13 DIVERSITY OF STRIATED MUSCLE 447 filaments. The gap is bridged by very thin (T) filaments. Micrograph, courtesy of A. Selverston. k FIG. 8. Low-power electron micrograph of longitudinal section through a single sarcomere of a unit of a B. nubilus lateral depressor stretched beyond the point of overlap of thick and thin quired by the theory that cross-bridges alone are responsible, have failed, even the most sophisticated and carefully-controlled ones (Gordon, Huxley, and Julian, 1966 a and b). Giant barnacle fibers stretched under load to twice their resting length about 30% beyond the overlap point will still contract extensively when depolarized by KC1. The forces developed at lengths beyond overlap are small, but they cannot be ignored. There is one last matter to which I would like to draw your attention. All of our ideas regarding the functioning of striated muscle derive from experiments on specialized fast muscle fibers of the frog. The ideas have never been adapted to explain slower and graded contractions which are of equal significance. It is not known what constitutes a graded contraction in micro-anatomical or molecular terms. Does it involve partial, but equal, activations of each sarcomere, or are individual sarcomeres activated to varying extents? We have attempted to answer these questions by rapid fixation for electron microscopy of fibers while stimulating them to develop graded contractions. The contractions were monitored by a tension recorder, and the membrane potential by an intracellular electrode. Most of the fibers treated in this way, from material as diverse as snake and barnacle, have shown similar appearances in the electron microscope. That is, individual sarcomeres, or even parts of half-sarcomeres, are heavily contracted, while others ranged through rest-length to heavily stretched (Fig. 6). To explain these findings I have proposed that the fundamental unit in excitationcontraction coupling is a single cisternal element of the SR (Hoyle, 1966). Either the cisternal elements or units (C. Us) have a range of thresholds or the amount of calcium released by each is directly proportional to the local potential difference (or current flow), or both possibilitites may occur. In a graded contraction only a proportion of the C. Us are excited, perhaps

14 448 GRAHAM HOYLE to different extents, resulting in a very heterogeneous set of microanatomical FIG. 9. Diagram to show proposed model of striated muscle structure including an elastic very thin (T) filament running the whole length of the sarcomere (and possibly the fiber). Actin and myosin filaments run parallel to the T filaments. changes but graded tension. A conventional "all-or-none" twitch would result from a rapid, nearly synchronous firing of all the C. Us. During the course of these investigations, I have found fibers which are in a state of partial (as much as 30%) contraction at normal resting potentials. These fibers give quick relaxations (negative twitches) in response to hyperpolarizing pulses. Other fibers have been found which contract in response to hyperpolarizing pulses of adequate strength, and yet others which contract strongly when a hyperpolarizing current is turned off, but not during its passage. None of the currently accepted models of excitation-contraction coupling can explain all of these diverse findings without qualifications. For comparative physiologists all of what I have said may have significance, though much of it will be controversial. It has long been clear, however, that the current orthodoxies regarding muscle ultrastructure as well as the hypothesis of the molecular basis of contraction and the mechanism of excitation-contraction coupling, should not be regarded as established without a great deal of further comparative study. Likewise, the notions of active state developed by the A.V. Hill school may need to be seriously questioned. If we can examine closely and come to fully understand some of the remarkable variety of ultrastructures and associated physiological function found in different muscles we are bound to illuminate our total understanding of this most important and fascinating tissue. REFERENCES Atwood, H. L., G. Hoyle, and T. Smyth, Jr Mechanical and electrical responses of single innervated crab-muscle fibers. J. Physiol. 180: Dorai-Raj, B. S Diversity of crab muscle fibers innervated by a single motor axon. J. Cell Comp. Physiol. 64: Garamvolgyi, N Observations preliminaires sur la structure de la strie Z dans le muscle alaire de l'abeille. J. Microscopie 2: Gauthier, G. F., and H. A. Padykula Cytological studies of fiber types in skeletal muscle. J. Cell Biol. 28: George, J. C, and N. M. G. Bhakthan

15 DIVERSITY OF STRIATED MUSCLE 449 Lipase activity in the slow- and fast-contracting leg muscles of the cockroach. Nature 192: 356. George, J. C, and A. K. Susheela, The histophysiological study of the rat diaphragm. Biol. Bull. 121: Ginsborg, B. L. I960. Some properties of avian skeletal muscle fibres with multiple neuromuscular junctions. J. Physiol. 154: Gordon, A. M., A. F. Huxley, and F. J. Julian, 1966a. Tension development in highly stretched vertebrate muscle fibers. J. Physiol. 184: Gordon, A. M., A. F. Huxley, and F. J. Julian. 1966&. The variation in isometric tension with sarcomere length in vertebrate muscle fibers. J. Physiol. 184: Hanson, J., and H. E. Huxley The structural basis of contraction in striated muscle. Symp. Soc. Exptl. Biol. 9: Hess, A The sarcoplasmic reticulum, the T system, and the motor terminals of slow and twitch muscle fibers in the garter snake. J. Cell Biol. 26: Hess, A., and G. Pilar, Slow fibres in the extraocular muscles of the cat. J. Physiol. 169: Hoyle, G Interpreting muscle function in invertebrates. Pfluger's Arch. 291: Hoyle, G., P. A. McXeill, and B. Walcott Nature of invaginating tubules in Felderstruktur muscle fibers of the garter snake. J. Cell Biol. 30: Huxley, H. E The mechanism of muscle contraction. Sci. Am. 213: Kuffler, S. W,. and E. M. Vaughan Williams Properties of the "slow" skeletal muscle fibres of the frog. J. Physiol. 121: Reedy, M In Discussion on the physical and chemical basis of muscular contraction. Proc. Roy. Soc. (London), B, 160:

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